Jiaheng Liu

Combined alkali and acid pretreatment of spent mushroom substrate for reducing sugar and biofertilizer production.

Spent mushroom substrate (SMS) was pretreated with alkaline reagents including potassium hydroxide, lime and ammonia to enhance enzymatic saccharification. Under the best pretreatment conditions (1M KOH, 80 °C, 90 min; 1M lime, 80 °C, 120 min; 10 M ammonia, 70 °C, 120 min), the total reducing sugar (TRS) yield reached 258.6, 204.2 and 251.2 mg/g raw SMS, which were respectively 6.15, 4.86, and 5.98 times of untreated SMS. The effects of pretreatment by above alkaline reagents and sulfuric acid on the composition and structure of SMS were evaluated to provide comparative performance data. A new process, combined alkali and acid (CAA) pretreatment followed by enzymatic hydrolysis, was innovatively proposed to improve the cost-effectiveness and avoid environmental problems. The SMS residue after CAA pretreatment-enzymatic hydrolysis process was converted to biofertilizer with Pichia farinose FL7 and a cell density of 3.0×10(8) cfu/g in biomass was attained.

Redox cofactor engineering in industrial microorganisms: strategies, recent applications and future directions

NAD and NADP, a pivotal class of cofactors, which function as essential electron donors or acceptors in all biological organisms, drive considerable catabolic and anabolic reactions. Furthermore, they play critical roles in maintaining intracellular redox homeostasis. However, many metabolic engineering efforts in industrial microorganisms towards modification or introduction of metabolic pathways, especially those involving consumption, generation or transformation of NAD/NADP, often induce fluctuations in redox state, which dramatically impede cellular metabolism, resulting in decreased growth performance and biosynthetic capacity. Here, we comprehensively review the cofactor engineering strategies for solving the problematic redox imbalance in metabolism modification, as well as their features, suitabilities and recent applications. Some representative examples of in vitro biocatalysis are also described. In addition, we briefly discuss how tools and methods from the field of synthetic biology can be applied for cofactor engineering. Finally, future directions and challenges for development of cofactor redox engineering are presented.

Improving xylose utilization of defatted rice bran for nisin production by overexpression of a xylose transcriptional regulator in Lactococcus lactis

Present investigation explores the potential of defatted rice bran (DRB) serving as sole carbon source and partial nitrogen source to support Lactococcus lactis growth and nisin production. To retain the nutrients in DRB, especially protein fractions, thermal pretreatment followed by enzymatic hydrolysis without washing step was applied for saccharification. A maximum of 45.64g reducing sugar mainly containing 30.26g glucose and 5.66g xylose from 100g DRB was attained in hydrolysates of DRB (HD). A novel strategy of xylR (xylose transcriptional regulator) overexpression followed by evolutionary engineering was proposed, which significantly increased the capacity of L. lactis to metabolize xylose. Subsequently, RT-PCR results indicated that xylR overexpression stimulated expression of xylose assimilation genes synergistically with exposure to xylose. In HD medium, the highest nisin titer of the engineered strain FEXR was 3824.53IU/mL, which was 1.37 times of that in sucrose medium by the original strain F44.

Improving nitrogen source utilization from defatted soybean meal for nisin production by enhancing proteolytic function of Lactococcus lactis F44

Nisin, one kind of natural antimicrobial peptide, is produced by certain Lactococcus lactis strains, which generally require expensive high-quality nitrogen sources due to limited ability of amino acids biosynthesis. Here we use defatted soybean meal (DSM) as sole nitrogen source to support L. lactis growth and nisin production. DSM medium composition and fermentation conditions were optimized using the methods of Plackett-Burman design and central composite design. The highest nisin production of 3879.58 IU/ml was obtained in DSM medium, which was 21.3% higher than that of commercial medium. To further increase the utilization ability of nitrogen sources, we enhanced the proteolytic function in L. lactis through rationally expressing the related enzymes, which were selected according to the compositions of amino acids and molecular weight of peptides in DSM medium. Significantly, an artificial proteolytic system consisting of a heterologous protease (NprB), an oligopeptides transporter subunit (OppA) and two peptidases (PepF and PepM) was introduced into L.lactis. The constructed strain BAFM was capable of achieving efficient biomass accumulation and nisin yield with 30% decreased amount of DSM hydrolysates, which further reduced the cost of nisin production. The strategy described here offers opportunities for low-cost L. lactis fermentation and large-scale nisin production in industry.

Global evolution of glycosylated polyene macrolide antibiotic biosynthesis

Antibiotics are the most marvelous evolutionary products of microbes to obtain competitive advantage and maintain ecological balance. However, the origination and development of antibiotics has yet to be explicitly investigated. Due to diverse structures and similar biosynthesis, glycosylated polyene macrolides (gPEMs) were chosen to explore antibiotic evolution. A total of 130 candidate and 38 transitional gPEM clusters were collected from actinomycetes genomes, providing abundant references for phenotypic gaps in gPEM evolution. The most conserved parts of gPEM biosynthesis were found and used for phylogeny construction. On this basis, we proposed ancestral gPEM clusters at different evolutionary stages and interpreted the possible evolutionary histories in detail. The results revealed that gPEMs evolved from small rings to large rings and continuously increased structural diversity through acquiring, discarding and exchanging genes from different evolutionary origins, as well as co-evolution of functionally related proteins. The combination of horizontal gene transfers, environmental effects and host preference resulted in the diversity and worldwide distribution of gPEMs. This study is not only a useful exploration on antibiotic evolution but also an inspiration for diversity and biogeographic investigations on antibiotics in the era of Big Data.

Two-stage carbon distribution and cofactor generation for improving l-threonine production of Escherichia coli: LIU et al.

L‐Threonine, a kind of essential amino acid, has numerous applications in food, pharmaceutical, and aquaculture industries. Fermentative l‐threonine production from glucose has been achieved in Escherichia coli. However, there are still several limiting factors hindering further improvement of l‐threonine productivity, such as the conflict between cell growth and production, byproduct accumulation, and insufficient availability of cofactors (adenosine triphosphate, NADH, and NADPH). Here, a metabolic modification strategy of two‐stage carbon distribution and cofactor generation was proposed to address the above challenges in E. coli THRD, an l‐threonine producing strain. The glycolytic fluxes towards tricarboxylic acid cycle were increased in growth stage through heterologous expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and citrate synthase, leading to improved glucose utilization and growth performance. In the production stage, the carbon flux was redirected into l‐threonine synthetic pathway via a synthetic genetic circuit. Meanwhile, to sustain the transaminase reaction for l‐threonine production, we developed an l‐glutamate and NADPH generation system through overexpression of glutamate dehydrogenase, formate dehydrogenase, and pyridine nucleotide transhydrogenase. This strategy not only exhibited 2.02‐ and 1.21‐fold increase in l‐threonine production in shake flask and bioreactor fermentation, respectively, but had potential to be applied in the production of many other desired oxaloacetate derivatives, especially those involving cofactor reactions.