Zhen Cai

Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco.

Photosynthetic CO2 fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first step of photosynthetic CO2 fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving photosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an efficient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coli-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G substitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxylation activity and a 45% improvement in catalytic efficiency towards CO2. The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.

From cyanochemicals to cyanofactories: a review and perspective

AbstractEngineering cyanobacteria for production of chemicals from solar energy, CO2 and water is a potential approach to address global energy and environment issues such as greenhouse effect. To date, more than 20 chemicals have been synthesized by engineered cyanobacteria using CO2 as raw materials, and these studies have been well reviewed. However, unlike heterotrophic microorganisms, the low CO2 fixation rate makes it a long way to go from cyanochemicals to cyanofactories. Here we review recent progresses on improvement of carbon fixation and redistribution of intercellular carbon flux, and discuss the challenges for developing cyanofactories in the future.

Genome replication engineering assisted continuous evolution (GREACE) to improve microbial tolerance for biofuels production.

BackgroundMicrobial production of biofuels requires robust cell growth and metabolism under tough conditions. Conventionally, such tolerance phenotypes were engineered through evolutionary engineering using the principle of “Mutagenesis followed-by Selection”. The iterative rounds of mutagenesis-selection and frequent manual interventions resulted in discontinuous and inefficient strain improvement processes. This work aimed to develop a more continuous and efficient evolutionary engineering method termed as “Genome Replication Engineering Assisted Continuous Evolution” (GREACE) using “Mutagenesis coupled-with Selection” as its core principle.ResultsThe core design of GREACE is to introduce an in vivo continuous mutagenesis mechanism into microbial cells by introducing a group of genetically modified proofreading elements of the DNA polymerase complex to accelerate the evolution process under stressful conditions. The genotype stability and phenotype heritability can be stably maintained once the genetically modified proofreading element is removed, thus scarless mutants with desired phenotypes can be obtained.Kanamycin resistance of E. coli was rapidly improved to confirm the concept and feasibility of GREACE. Intrinsic mechanism analysis revealed that during the continuous evolution process, the accumulation of genetically modified proofreading elements with mutator activities endowed the host cells with enhanced adaptation advantages. We further showed that GREACE can also be applied to engineer n-butanol and acetate tolerances. In less than a month, an E. coli strain capable of growing under an n-butanol concentration of 1.25% was isolated. As for acetate tolerance, cell growth of the evolved E. coli strain increased by 8-fold under 0.1% of acetate. In addition, we discovered that adaptation to specific stresses prefers accumulation of genetically modified elements with specific mutator strengths.ConclusionsWe developed a novel GREACE method using “Mutagenesis coupled-with Selection” as core principle. Successful isolation of E. coli strains with improved n-butanol and acetate tolerances demonstrated the potential of GREACE as a promising method for strain improvement in biofuels production.

Engineering cellular robustness of microbes by introducing the GroESL chaperonins from extremophilic bacteria

The cellular robustness is a big concern for efficient microbial production of biofuels and biochemicals. In this study, the groESL genes from extremophilic bacteria were found to serve as transplantable stress-response elements to improve diverse types of stress-tolerances of other microbes. By overexpressing the groESL from the solvent-tolerant Pseudomonas putida in Escherichia coli, its thermo-tolerance and ethanol-tolerance were significantly increased. Meanwhile, the groESL from the thermophilic Thermoanaerobacter tengcongensis endowed Clostridium acetobutylicum with improved corn cob hydrolysates (CCH)-tolerance as well as elevated butanol productivity. The chaperonins GroESL have been widely considered as cellular stress-response proteins and overexpression of native groESL has been proven to improve cellular tolerances facing various stresses. Here we found that the groESL genes from extremophilic bacteria were superior to the native ones, possibly because they have adapted to the environmental stresses during long-term natural evolution. Moreover, our results also revealed that different extreme groESL genes performed quite different in different microbes. Thus the relation and compatibility between the extremophiles and the host must be considered for selection of the proper groESL for engineering microbial robustness.

Design and Construction of a Non-Natural Malate to 1,2,4-Butanetriol Pathway Creates Possibility to Produce 1,2,4-Butanetriol from Glucose

1,2,4-butanetriol (BT) is an important bulk chemical mainly used for producing the superior energetic plasticizer (1,2,4-butanetriol trinitrate) in propellant and explosive formulations. BT is commercially produced by chemical synthesis from petroleum-based feedstocks; until recently a costly biosynthetic route from xylose or arabinose was reported. Here we designed a novel biosynthetic pathway for BT from malate, for the purpose of using glucose as an alternative and cheaper substrate in future. This biosynthetic pathway was achieved through six sequential enzymatic reactions. Following tests of several combinations of enzymes for the pathway, five enzymes including malate thiokinase, succinate-semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate CoA-transferase and bifunctional aldehyde/alcohol dehydrogenase were finally chosen. All enzyme genes were expressed on two compatible plasmids in E. coli, and their functions verified separately. Following assembly of two functional modules, BT was detected in the fermentation broth upon addition of malate, proving BT can be biosynthesized from malate. Furthermore, BT was detected in the fermentation using glucose as the sole carbon source, suggesting that such novel BT biosynthetic pathway has created the possibility for the production of BT from the cheaper substrate glucose.

Engineering stress tolerance of Escherichia coli by stress‐induced mutagenesis (SIM)‐based adaptive evolution

Microbial tolerance to toxic products and biomass hydrolysates is a challenge for the production of fuels and chemicals from renewable resources. To improve cellular tolerance to these environmental stresses, a novel adaptive evolutionary strategy based on stress‐induced mutagenesis (SIM) was developed using non‐dividing cells. The concept of this method was proved using Escherichia coli FC40 as a model strain, which was used to quantitatively evaluate the rate of SIM. By deleting either the mutL or mutS gene to disturb the mismatch repair activity of the host cells, the SIM rate under stressful conditions increased by 92‐ and 57‐fold, respectively. A periodic SIM‐based adaptive evolution procedure, which synchronized the mutagenesis and the selection process in a single plate‐incubation step, was then developed using the mutL‐deleted mutant. E. coli mutants tolerant to high concentrations of butanol (13 g/L), NaCl (95 g/L), and high temperature (50°C) were obtained. These results indicate that stress‐induced adaptive evolution in non‐dividing cells is an effective approach that can improve microbial tolerance against various stresses and generate robust microbial strains suitable for production of fuels and chemicals.

Development of a stress-induced mutagenesis module for autonomous adaptive evolution of Escherichia coli to improve its stress tolerance

BackgroundMicrobial tolerance to different environmental stresses is of importance for efficient production of biofuels and biochemical. Such traits are often improved by evolutionary engineering approaches including mutagen-induced mutagenesis and successive passage. In contrast to these approaches which generate mutations in rapidly growing cells, recent research showed that mutations could be generated in non-dividing cells under stressful but non-lethal conditions, leading to the birth of the theory of stress-induced mutagenesis (SIM). A molecular mechanism of SIM has been elucidated to be mutagenic repair of DNA breaks. This inspired us to develop a synthetic SIM module to simulate the mutagenic cellular response so as to accelerate microbial adaptive evolution for an improved stress tolerance.ResultsA controllable SIM evolution module was devised based on a genetic toggle switch in Escherichia coli. The synthetic module enables expression and repression of the genes related to up- and down-regulation responses during SIM in a bistable way. Upon addition of different inducers, the module can be turned on or off, triggering transition to a mutagenic or a high-fidelity state and thus allowing periodic adaptive evolution. Six genes (recA, dinB, umuD, ropS, ropE, and nusA) in the up-regulation responses were evaluated for their potentials to enhance the SIM rate. Expression of recA, dinB, or ropS alone increased the SIM rate by 4.5- to 13.7-fold, whereas their combined expression improved the rate by 31.9-fold. Besides, deletion of mutL increased the SIM rate by 82-fold. Assembly of these genes into the SIM module in the mutL-deletion E. coli strain elevated the SIM rate by nearly 3000-fold. Accelerated adaptive evolution of E. coli equipped with this synthetic SIM module was demonstrated under n-butanol stress, with the minimal inhibitory concentration of n-butanol increasing by 56 % within 2.5 months.ConclusionsA synthetic SIM module was constructed to simulate cellular mutagenic responses during SIM. Based on this, a novel evolutionary engineering approach—SIM-based adaptive evolution—was developed to improve the n-butanol tolerance of E. coli.

Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors

Engineering the cofactor specificity of a natural enzyme often results in a significant decrease in its activity on original cofactor. Here we report that a NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842 can be rationally engineered to efficiently use both NADH and NADPH as cofactors. Point mutations on three amino acids (D176S, I177R, F178T) predicted by computational analysis resulted in a modified enzyme designated as d-LDH*. The Kcat/Km of the purified d-LDH* on NADPH increased approximately 184-fold while the Kcat/Km on NADH also significantly increased, showing for the first time that a rationally engineered d-LDH could exhibit comparable activity on both NADPH and NADH. Further kinetic analysis revealed that the enhanced affinity with NADH or NADPH and the significant increased Kcat of d-LDH* resulted in the significant increase of d-LDH* activity on both NADPH and NADH. This study thus demonstrated that the cofactor specificity of dehydrogenase can be broadened by using targeted engineering approach, and the engineered enzyme can efficiently function in NADH-rich, or NADPH-rich, or NADH and NADPH-rich environment.

Synthetic biology for CO2 fixation

Recycling of carbon dioxide (CO2) into fuels and chemicals is a potential approach to reduce CO2 emission and fossil-fuel consumption. Autotrophic microbes can utilize energy from light, hydrogen, or sulfur to assimilate atmospheric CO2 into organic compounds at ambient temperature and pressure. This provides a feasible way for biological production of fuels and chemicals from CO2 under normal conditions. Recently great progress has been made in this research area, and dozens of CO2-derived fuels and chemicals have been reported to be synthesized by autotrophic microbes. This is accompanied by investigations into natural CO2-fixation pathways and the rapid development of new technologies in synthetic biology. This review first summarizes the six natural CO2-fixation pathways reported to date, followed by an overview of recent progress in the design and engineering of CO2-fixation pathways as well as energy supply patterns using the concept and tools of synthetic biology. Finally, we will discuss future prospects in biological fixation of CO2.

Comparative genome analysis of a thermotolerant Escherichia coli obtained by Genome Replication Engineering Assisted Continuous Evolution (GREACE) and its parent strain provides new understanding of microbial heat tolerance

Heat tolerance of microbes is of great importance for efficient biorefinery and bioconversion. However, engineering and understanding of microbial heat tolerance are difficult and insufficient because it is a complex physiological trait which probably correlates with all gene functions, genetic regulations, and cellular metabolisms and activities. In this work, a novel strain engineering approach named Genome Replication Engineering Assisted Continuous Evolution (GREACE) was employed to improve the heat tolerance of Escherichia coli. When the E. coli strain carrying a mutator was cultivated under gradually increasing temperature, genome-wide mutations were continuously generated during genome replication and the mutated strains with improved thermotolerance were autonomously selected. A thermotolerant strain HR50 capable of growing at 50°C on LB agar plate was obtained within two months, demonstrating the efficiency of GREACE in improving such a complex physiological trait. To understand the improved heat tolerance, genomes of HR50 and its wildtype strain DH5α were sequenced. Evenly distributed 361 mutations covering all mutation types were found in HR50. Closed material transportations, loose genome conformation, and possibly altered cell wall structure and transcription pattern were the main differences of HR50 compared with DH5α, which were speculated to be responsible for the improved heat tolerance. This work not only expanding our understanding of microbial heat tolerance, but also emphasizing that the in vivo continuous genome mutagenesis method, GREACE, is efficient in improving microbial complex physiological trait.