Key role of K+ and Ca2+ in high-yield ethanol production by S. Cerevisiae from concentrated sugarcane molasses.

BACKGROUND: Saccharomyces cerevisiae is an important microorganism in ethanol synthesis, and with sugarcane molasses as the feedstock, ethanol is being synthesized sustainably to meet growing demands. However, high-concentration ethanol fermentation based on high-concentration sugarcane molasses-which is needed for reduced energy consumption of ethanol distillation at industrial scale-is yet to be achieved. RESULTS: In the present study, to identify the main limiting factors of this process, adaptive laboratory evolution and high-throughput screening (Py-Fe3+) based on ARTP (atmospheric and room-temperature plasma) mutagenesis were applied. We identified high osmotic pressure, high temperature, high alcohol levels, and high concentrations of K+, Ca2+, K+ and Ca2+ (K+&Ca2+), and sugarcane molasses as the main limiting factors. The robust S. cerevisiae strains of NGT-F1, NGW-F1, NGC-F1, NGK+, NGCa2+ NGK+&Ca2+-F1, and NGTM-F1 exhibited high tolerance to the respective limiting factor and exhibited increased yield. Subsequently, ethanol synthesis, cell morphology, comparative genomics, and gene ontology (GO) enrichment analysis were performed in a molasses broth containing 250 g/L total fermentable sugars (TFS). Additionally, S. cerevisiae NGTM-F1 was used with 250 g/L (TFS) sugarcane molasses to synthesize ethanol in a 5-L fermenter, giving a yield of 111.65 g/L, the conversion of sugar to alcohol reached 95.53%. It is the highest level of physical mutagenesis yield at present. CONCLUSION: Our results showed that K+ and Ca2+ ions primarily limited the efficient production of ethanol. Then, subsequent comparative transcriptomic GO and pathway analyses showed that the co-presence of K+ and Ca2+ exerted the most prominent limitation on efficient ethanol production. The results of this study might prove useful by promoting the development and utilization of green fuel bio-manufactured from molasses.

Enhancing the Production of Pinene in Escherichia coli by Using a Combination of Shotgun, Product-Tolerance and I-SceI Cleavage Systems.

Tolerance breeding through genetic engineering, sequence and omics analyses, and gene identification processes are widely used to synthesize biofuels. The majority of related mechanisms have been shown to yield endogenous genes with high expression. However, the process was time-consuming and labor-intensive, meaning there is a need to address the problems associated with the low-throughput screening method and significant time and money consumption. In this study, a combination of the limit screening method (LMS method) and product-tolerance engineering was proposed and applied. The Escherichia coli MG1655 genomic DNA library was constructed using the shotgun method. Then, the cultures were incubated at concentrations of 0.25%, 0.5%, 0.75% and 1.0% of pinene with different inhibitory effects. Finally, the genes acrB, flgFG, motB and ndk were found to be associated with the enhanced tolerance of E. coli to pinene. Using the I-SceI cleavage system, the promoters of acrB, flgFG and ndk genes were replaced with P37. The final strain increased the production of pinene from glucose by 2.1 times.

Biosensor-assisted evolution for high-level production of 4-hydroxyphenylacetic acid in Escherichia coli.

4-Hydroxyphenylacetic acid (4HPAA) is an important building block for synthesizing drugs, agrochemicals, and biochemicals, and requires sustainable production to meet increasing demand. Here, we use a 4HPAA biosensor to overcome the difficulty of conventional library screening in identification of preferred mutants. Strains with higher 4HPAA production and tolerance are successfully obtained by atmospheric and room temperature plasma (ARTP) mutagenesis coupled with adaptive laboratory evolution using this biosensor. Genome shuffling integrates preferred properties in the strain GS-2-4, which produces 25.42 g/L 4HPAA. Chromosomal mutations of the strain GS-2-4 are identified by whole genome sequencing. Through comprehensive analysis and experimental validation, important genes, pathways and regulations are revealed. The best gene combination in inverse engineering, acrD-aroG, increases 4HPAA production of strain GS-2-4 by 37% further. These results emphasize precursor supply and stress resistance are keys to efficient 4HPAA biosynthesis. Our work shows the power of biosensor-assisted screening of mutants from libraries. The methods developed here can be easily adapted to construct cell factories for the production of other aromatic chemicals. Our work also provides many valuable target genes to build cell factories for efficient 4HPAA production in the future.

Enhanced Production of Pterostilbene in Escherichia coli Through Directed Evolution and Host Strain Engineering.

Pterostilbene is a derivative of resveratrol with a higher bioavailability and biological activity, which shows antioxidant, anti-inflammatory, antitumor, and antiaging activities. Here, directed evolution and host strain engineering were used to improve the production of pterostilbene in Escherichia coli. First, the heterologous biosynthetic pathway enzymes of pterostilbene, including tyrosine ammonia lyase, p-coumarate: CoA ligase, stilbene synthase, and resveratrol O-methyltransferase, were successively directly evolved through error-prone polymerase chain reaction (PCR). Four mutant enzymes with higher activities of in vivo and in vitro were obtained. The directed evolution of the pathway enzymes increased the pterostilbene production by 13.7-fold. Then, a biosensor-guided genome shuffling strategy was used to improve the availability of the precursor L-tyrosine of the host strain E. coli TYR-30 used for the production of pterostilbene. A shuffled E. coli strain with higher L-tyrosine production was obtained. The shuffled strain harboring the evolved pathway produced 80.04 ± 5.58 mg/l pterostilbene, which is about 2.3-fold the highest titer reported in literatures.

Cell-free Biosynthesis of Chlorogenic Acid Using a Mixture of Chassis Cell Extracts and Purified Spy-Cyclized Enzymes.

A novel cell-free biosynthesis system based on a mixture of chassis cell extracts and purified Spy-cyclized enzymes (CFBS-mixture) was developed. As a demonstration, the CFBS-mixture was applied to chlorogenic acid (CGA) biosynthesis. The mix-and-match and Plackett-Burman experiments demonstrated that Lonicera japonica hydroxycinnamate-CoA quinate transferase and p-hydroxyphenylacetate 3-hydroxylase were the key enzymes for the production of CGA. After optimization of the concentrations of the biosynthetic enzymes in the CFBS-mixture reaction using the Plackett-Burman experimental design and the path of the steepest ascent, 711.26 ± 15.63 mg/L CGA was produced after 16 h, which is 71.1-fold the yield obtained using the conventional crude extract-based CFBS and 9.1-fold the reported yield obtained using the living cells. Based on the CFBS-mixture results, the production of CGA was further enhanced in engineered Escherichia coli. The CFBS-mixture strategy is highly effective and will be useful for high-level CFBS of natural products.

Biosensor-Guided Atmospheric and Room-Temperature Plasma Mutagenesis and Shuffling for High-Level Production of Shikimic Acid from Sucrose in Escherichia coli.

Here, we first developed a combined strain improvement strategy of biosensor-guided atmospheric and room-temperature plasma mutagenesis and genome shuffling. Application of this strategy resulted in a 2.7-fold increase in the production of shikimic acid (SA) and a 2.0-fold increase in growth relative to those of the starting strain. Whole-cell resequencing of the shuffled strain and confirmation using CRISPRa/CRISPRi revealed that some membrane protein-related mutant genes are identified as being closely related to the higher SA titer. The engineered shuffling strain produced 18.58 ± 0.56 g/L SA from glucose with a yield of 68% (mol/mol) by fed-batch whole-cell biocatalysis, achieving 79% of the theoretical maximum. Sucrose-utilizing Escherichia coli was engineered for SA production by introducing Mannheimia succiniciproducens ß-fructofuranosidase gene. The resulting sucrose-utilizing E. coli strain produced 24.64 ± 0.32 g/L SA from sucrose with a yield of 1.42 mol/mol by fed-batch whole-cell biocatalysis, achieving 83% of the theoretical maximum.

Recent Advances in Metabolically Engineered Microorganisms for the Production of Aromatic Chemicals Derived From Aromatic Amino Acids.

Aromatic compounds derived from aromatic amino acids are an important class of diverse chemicals with a wide range of industrial and commercial applications. They are currently produced via petrochemical processes, which are not sustainable and eco-friendly. In the past decades, significant progress has been made in the construction of microbial cell factories capable of effectively converting renewable carbon sources into value-added aromatics. Here, we systematically and comprehensively review the recent advancements in metabolic engineering and synthetic biology in the microbial production of aromatic amino acid derivatives, stilbenes, and benzylisoquinoline alkaloids. The future outlook concerning the engineering of microbial cell factories for the production of aromatic compounds is also discussed.

Enhanced Production of Pinene by Using a Cell-Free System with Modular Cocatalysis.

α-Pinene is an important monoterpene that is widely used as a pharmaceutical product, biofuel, and so forth. We first established a cell-free system with modular cocatalysis for the production of pinene from glucose. After optimization of the compositions of the cell-free reaction mixture using the Plackett-Burman experimental design and the path of steepest ascent, the production of pinene increased by 57%. It was found that ammonium acetate, NAD+, and NADPH are the three most important parameters for the production of pinene. Mix-and-match experiments showed that the simultaneous addition of the lysate of Escherichia coli overexpressing native 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, SufBCD Fe-S cluster assembly protein, isopentenyl-diphosphate isomerase, and Pinus taeda pinene synthase improved the production of pinene. Increasing the enzyme concentration of the extract further enhanced the production of pinene to 1256.31 ± 46.12 mg/L with a productivity of 104.7 mg/L h, almost 1.2-fold faster than any system reported thus far. This study demonstrates that a cell-free system is a powerful and robust platform for biomanufacture.

Genomic and transcriptional changes in response to pinene tolerance and overproduction in evolved Escherichia coli.

α-Pinene is an important monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals and biofuels. Although an evolved strain Escherichia coli YZFP, which had higher tolerance to pinene and titer, has been successfully used to produce high levels of pinene, the pinene titer is much lower than that of hemiterpene (isoprene) and sesquiterpenes (farnesene) to date. Moreover, the overall cellular physiological and metabolic changes caused by higher tolerance to pinene and overproduction of pinene remains unclear. To reveal the mechanism of Escherichia coli YZFP with the higher tolerance to pinene and titer, a comparative genomics and transcriptional level analyses combining with CRISPR activation (CRISPRa) and interference (CRISPRi) were carried out. The results show that the tolerance to pinene and the overproduction of pinene in E. coli may be associated with: 1) the mutations of the DXP pathway genes, the rpoA and some membrane protein genes, and their upregulations of transcription levels; and 2) the mutations of some genes and their downregulation of transcriptional levels. These comparative omics analyses provided some genetic modification strategies to further improve pinene production. Overexpression of the mutated cbpA, tabA, pitA, rpoA, sufBCDS, mutS, ispH, oppF, dusB, dnaK, dxs, dxr and flgFGH genes further improved pinene production. This study also demonstrated that combining comparative omics analysis with CRISPRa and CRISPRi is an efficient technology to quickly find a new metabolic engineering strategy.

Enhancing Production of Pinene in Escherichia coli by Using a Combination of Tolerance, Evolution, and Modular Co-culture Engineering.

α-Pinene is a natural and active monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals, and biofuels. Although it has been successfully produced by genetically engineered microorganisms, the production level of pinene is much lower than that of hemiterpene (isoprene) and sesquiterpenes (farnesene) to date. We first improved pinene tolerance to 2.0% and pinene production by adaptive laboratory evolution after atmospheric and room temperature plasma (ARTP) mutagenesis and overexpression of the efflux pump to obtain the pinene tolerant strain Escherichia coli YZFP, which is resistant to fosmidomycin. Through error-prone PCR and DNA shuffling, we isolated an Abies grandis geranyl pyrophosphate synthase variant that outperformed the wild-type enzyme. To balance the expression of multiple genes, a tunable intergenic region (TIGR) was inserted between A. grandis GPPSD90G/L175P and Pinus taeda Pt1Q457L . In an effort to improve the production, an E. coli-E. coli modular co-culture system was engineered to modularize the heterologous mevalonate (MEV) pathway and the TIGR-mediated gene cluster of A. grandis GPPSD90G/L175P and P. taeda Pt1Q457L . Specifically, the MEV pathway and the TIGR-mediated gene cluster were integrated into the chromosome of the pinene tolerance strain E. coli YZFP and then evolved to a higher gene copy number by chemically induced chromosomal evolution, respectively. The best E. coli-E. coli co-culture system of fermentation was found to improve pinene production by 1.9-fold compared to the mono-culture approach. The E. coli-E. coli modular co-culture system of whole-cell biocatalysis further improved pinene production to 166.5 mg/L.

Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-based biofuels.

Isoprenoids are the most abundant and highly diverse group of natural products. Many isoprenoids have been used for pharmaceuticals, nutraceuticals, flavors, cosmetics, food additives and biofuels. Carotenoids and isoprenoid-based biofuels are two classes of important isoprenoids. These isoprenoids have been produced microbially through metabolic engineering and synthetic biology efforts. Herein, we briefly review the engineered biosynthetic pathways in well-characterized microbial systems for the production of carotenoids and several isoprenoid-based biofuels.