Xiaomei Lv

Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae

Balanced utilization of metabolic intermediates and controllable expression of genes in biosynthetic pathways are key issues for the effective production of value-added chemicals in microbes. An inducer/repressor-free sequential control strategy regulated by glucose concentration in the growth environment was proposed to address these issues, and its efficiency was validated using heterologous beta-carotenoid biosynthesis in Saccharomyces cerevisiae as an example. Through sequential control of the downstream, upstream, and competitive pathways of farnesyl diphosphate (FPP), the crucial metabolic node in the biosynthesis of terpenoids, in a predetermined order, a carotenoid production of 1156 mg/L (20.79 mg/g DCW) was achieved by high-cell density fermentation. Quantitative PCR analysis of the regulated genes demonstrated that the transcription patterns were controlled in a sequential manner as expected. The inducer/repressor-free nature of this strategy offers a both practical and economically efficient approach to improved biosynthetic production of value-added chemicals.

Construction of a controllable β‐carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an important platform organism for the synthesis of a great number of natural products. However, the assembly of controllable and genetically stable heterogeneous biosynthetic pathways in S. cerevisiae still remains a significant challenge. Here, we present a strategy for reconstructing controllable multi‐gene pathways by employing the GAL regulatory system. A set of marker recyclable integrative plasmids (pMRI) was designed for decentralized assembly of pathways. As proof‐of‐principle, a controllable β‐carotene biosynthesis pathway (∼16 kb) was reconstructed and optimized by repeatedly using GAL10–GAL1 bidirectional promoters with high efficiency (80–100%). By controling the switch time of the pathway, production of 11 mg/g DCW of total carotenoids (72.57 mg/L) and 7.41 mg/g DCW of β‐carotene was achieved in shake‐flask culture. In addition, the engineered yeast strain exhibited high genetic stability after 20 generations of subculture. The results demonstrated a controllable and genetically stable biosynthetic pathway capable of increasing the yield of target products. Furthermore, the strategy presented in this study could be extended to construct other pathways in S. cerevisisae. Biotechnol. Bioeng. 2014;111: 125–133.

Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering.

Improved supply of farnesyl diphosphate (FPP) is often considered as a typical strategy for engineering Saccharomyces cerevisiae towards efficient terpenoid production. However, in the engineered strains with enhanced precursor supply, the production of the target metabolite is often impeded by insufficient capacity of the heterologous terpenoid pathways, which limits further conversion of FPP. Here, we tried to assemble an unimpeded biosynthesis pathway by combining directed evolution and metabolic engineering in S. cerevisiae for lycopene-overproduction. First, the catalytic ability of phytoene syntheses from different sources was investigated based on lycopene accumulation. Particularly, the lycopene cyclase function of the bifunctional enzyme CrtYB from Xanthophyllomyces dendrorhous was inactivated by deletion of functional domain and directed evolution to obtain mutants with solely phytoene synthase function. Coexpression of the resulting CrtYB11M mutant along with the CrtE and CrtI genes from X. dendrorhous, and the tHMG1 gene from S. cerevisiae led to production of 4.47 mg/g DCW (Dry cell weight) of lycopene and 25.66 mg/g DCW of the by-product squalene. To further increase the FPP competitiveness of the lycopene synthesis pathway, we tried to enhance the catalytic performance of CrtE by directed evolution and created a series of pathway variants by varying the copy number of Crt genes. Finally, fed-batch fermentation was conducted for the diploid strain YXWPD-14 resulting in accumulation of 1.61 g/L (24.41 mg/g DCW) of lycopene, meanwhile, the by-production of squalene was reduced to below 1 mg/g DCW.

Enhanced isoprene biosynthesis in Saccharomyces cerevisiae by engineering of the native acetyl-CoA and mevalonic acid pathways with a push-pull-restrain strategy

To explore the capacity of isoprene production in Saccharomyces cerevisiae, a rational push-pull-restrain strategy was proposed to engineer the mevalonic acid (MVA) and acetyl-CoA pathways. The strategy can be decomposed into the up-regulation of precursor supply in the acetyl-CoA module and the MVA pathway (push-strategy), increase of the isoprene branch flux (pull-strategy), and down-regulation of the competing pathway (restrain-strategy). Furthermore, to reduce the production cost arising from galactose addition and meanwhile maintain the high expression of Gal promoters, the galactose regulatory network was modulated by Gal80p deletion. Finally, the engineered strain YXM10-ispS-ispS could accumulate up to 37 mg/L isoprene (about 782-fold increase compared to the parental strain) under aerobic conditions with glycerol-sucrose as carbon source. In this way, a new potential platform for isoprene production was established via metabolic engineering of the yeast native pathways.

Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae

Microbial production of isoprene from renewable feedstock is a promising alternative to traditional petroleum-based processes. Currently, efforts to improve isoprenoid production in Saccharomyces cerevisiae mainly focus on cytoplasmic engineering, whereas comprehensive engineering of multiple subcellular compartments is rarely reported. Here, we propose dual metabolic engineering of cytoplasmic and mitochondrial acetyl-CoA utilization to boost isoprene synthesis in S. cerevisiae. This strategy increases isoprene production by 2.1-fold and 1.6-fold relative to the recombinant strains with solely mitochondrial or cytoplasmic engineering, respectively. By combining a modified reiterative recombination system for rapid pathway assembly, a two-phase culture process for dynamic metabolic regulation, and aerobic fed-batch fermentation for sufficient supply of acetyl-coA and carbon, we achieve 2527, mg l−1 of isoprene, which is the highest ever reported in engineered eukaryotes. We propose this strategy as an efficient approach to enhancing isoprene production in yeast, which might open new possibilities for bioproduction of other value-added chemicals.

Combining Gal4p-mediated expression enhancement and directed evolution of isoprene synthase to improve isoprene production in Saccharomyces cerevisiae

Current studies on microbial isoprene biosynthesis have mostly focused on regulation of the upstream mevalonic acid (MVA) or methyl-erythritol-4-phosphate (MEP) pathway. However, the downstream bottleneck restricting isoprene biosynthesis capacity caused by the weak expression and low activity of plant isoprene synthase (ISPS) under microbial fermentation conditions remains to be alleviated. Here, based on a previously constructed Saccharomyces cerevisiae strain with enhanced precursor supply, we strengthened the downstream pathway through increasing both the expression and activity of ISPS to further improve isoprene production. Firstly, a two-level expression enhancement system was developed for the PGAL1-controlled ISPS by overexpression of GAL 4. Meanwhile, the native GAL1/7/10 promoters were deleted to avoid competition for the transcriptional activator Gal4p, and GAL80 was disrupted to eliminate the dependency of gene expression on galactose induction. The IspS expression was obviously elevated upon enhanced Gal4p supply, and the isoprene production was improved from 6.0mg/L to 23.6mg/L in sealed-vial cultures with sucrose as carbon source. Subsequently, a novel high-throughput screening method was developed based on precursor toxicity and used for ISPS directed evolution towards enhanced catalytic activity. Combinatorial mutagenesis of the resulting ISPS mutants generated the best mutant ISPSM4, introduction of which into the GAL4-overexpressing strain YXM29 achieved 50.2mg/L of isoprene in sealed vials, and the isoprene production reached 640mg/L and 3.7g/L in aerobic batch and fed-batch fermentations, respectively. These results demonstrated the effectiveness of the proposed combinatorial engineering strategy in isoprene biosynthesis, which might also be feasible and instructive for biotechnological production of other valuable chemicals.

Engineering microbes for isoprene production

Isoprene is facing a growing global market due to its wide industrial applications. Current industrial production of isoprene is almost entirely petroleum-based, which is influenced by the shrinking C5 supply, while the natural emission of isoprene is predominantly contributed by plants. To bridge the need gap, a highly efficient fermentation-based process for isoprene production might be a suitable and sustainable solution, and extensive research works have been performed to achieve this goal. Here we review the accomplishments in this field by summarizing the history and prospects of microbial isoprene production. The natural producers and biosynthesis pathways of isoprene, the key enzyme isoprene synthase and the metabolic engineering strategies adopted for developing isoprene-producing microorganisms are introduced. In particular, strategies employed for achieving engineered strains with improved performance indices are discussed based on the published papers and patents. The perspectives on further performance improvements and potential future strategies are presented as well.

Combinatorial pathway optimization in Escherichia coli by directed co-evolution of rate-limiting enzymes and modular pathway engineering.

Metabolic engineering of microorganisms for heterologous biosynthesis is a promising route to sustainable chemical production which attracts increasing research and industrial interest. However, the efficiency of microbial biosynthesis is often restricted by insufficient activity of pathway enzymes and unbalanced utilization of metabolic intermediates. This work presents a combinatorial strategy integrating modification of multiple rate‐limiting enzymes and modular pathway engineering to simultaneously improve intra‐ and inter‐pathway balance, which might be applicable for a range of products, using isoprene as an example product. For intra‐module engineering within the methylerythritol‐phosphate (MEP) pathway, directed co‐evolution of DXS/DXR/IDI was performed adopting a lycopene‐indicated high‐throughput screening method developed herein, leading to 60% improvement of isoprene production. In addition, inter‐module engineering between the upstream MEP pathway and the downstream isoprene‐forming pathway was conducted via promoter manipulation, which further increased isoprene production by 2.94‐fold compared to the recombinant strain with solely protein engineering and 4.7‐fold compared to the control strain containing wild‐type enzymes. These results demonstrated the potential of pathway optimization in isoprene overproduction as well as the effectiveness of combining metabolic regulation and protein engineering in improvement of microbial biosynthesis. Biotechnol. Bioeng. 2016;113: 2661–2669.

Directed evolution of an exoglucanase facilitated by a co-expressed β-glucosidase and construction of a whole engineered cellulase system in Escherichia coli

A novel high-throughput screening method is proposed for the directed evolution of exoglucanase facilitated by the co-expression of β-glucosidase, using the glucose released from filter paper as the screening indicator. Three transformants (B1, D6 and G10) with improved activity were selected from 4,000 colonies. The specific activities of B1, D6 and G10 for releasing glucose were, respectively, 1.4-, 1.3- and 1.6-fold higher than that of the wild type. The engineered exoglucanase gene was inserted into an expression vector carrying the previously engineered endoglucanase and β-glucosidase genes, and transformed into Escherichia coli to form a completely engineered cellulase system that showed 8.2-fold increase in glucose production (relative activity) compared to the cells equipped with wild-type enzymes. To our knowledge, this is the first report for directed evolution of an exoglucanase using insoluble cellulose as the screening substrate.

Identification and elimination of metabolic bottlenecks in the quinone modification pathway for enhanced coenzyme Q10 production in Rhodobacter sphaeroides

In this report, UbiE and UbiH in the quinone modification pathway (QMP) were identified in addition to UbiG as bottleneck enzymes in the CoQ10 biosynthesis by Rhodobacter sphaeroides. The CoQ10 content was enhanced after co-overexpression of UbiE and UbiG, however, accompanied by the accumulation of the intermediate 10P-MMBQ. UbiH was then co-overexpressed to pull the metabolic flux towards downstream, resulting in an elevated CoQ10 productivity and decreased biomass. On the other hand, the expression levels of UbiE and UbiG were tuned to eliminate the intermediate accumulation, however at the sacrifice of productivity. To alleviate the detrimental effect on either productivity or cell growth, we tried to fuse UbiG with UbiE and localize them onto the membrane to elevate intermediate conversion. By fusing UbiE and UbiG to pufX, CoQ10 was accumulated to 108.51±2.76mg/L with a biomass of 12.2±0.9g/L. At last, we combined the optimized QMP and the previously engineered 2-methyl-d-erythritol-4-phosphate pathway (MEP) to further boost CoQ10 biosynthesis, resulting in a strain with 138±2.64mg/L CoQ10 production.