Indole-3-acetic acid (IAA) is a critical plant hormone that regulates cell division, development, and metabolism. IAA synthesis in plants and plant-associated microorganisms cannot fulfill the requirement for large-scale agricultural production. Here, two novel IAA biosynthesis pathways, tryptamine (TAM) and indole-3-acetamide (IAM), were developed for IAA production by whole-cell catalysis and de novo biosynthesis in an engineered Escherichia coli MG1655. When 10 g/L L-tryptophan was used as a substrate, an MIA-6 strain containing a heterologous IAM pathway had the highest IAA titer of 7.10 g/L (1.34 × 10 3 mg/g DCW), which was 98.4 times more than MTAI-5 containing the TAM pathway by whole-cell catalysis. De novo IAA biosynthesis was optimized by improving NAD(P)H availability, resulting in an increased IAA titer of 906 mg/L obtained by the MGΔadhE::icd strain, which is 29.7% higher than the control. These strategies exhibit the potential for IAA production in engineered E. coli and possible industrial applications.
BackgroundThe ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in T. reesei. Currently, only the locus of exocellobiohydrolases I encoding gene (cbh1) is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized.ResultsTo search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an Aspergillus niger lipA gene and Discosoma sp. DsRed1 gene in T. reesei, by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to cbh1 gene locus, two other genome insertion loci that efficiently facilitate gene expression in T. reesei were identified.ConclusionsWe have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in T. reesei. Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1139-3) contains supplementary material, which is available to authorized users.
Background: In this study, renewable tea waste hydrolysate was used as a sole carbon source for carotenoids and lipid production. A novel Rhodosporidium toruloides mutant strain, RM18, was isolated through atmospheric and room-temperature plasma mutagenesis and continuous domestication in tea waste hydrolysate from R. toruloides ACCC20341.Results: RM18 produced a larger biomass and more carotenoids and α-linolenic acid compared with the control strain cultured in tea waste hydrolysate. The highest yields of torularhodin (481.92 μg/g DCW) and torulene (501 μg/g DCW) from RM18 cultured in tea waste hydrolysate were 12.86-and 1.5-fold higher, respectively, than that of the control strain. In addition, α-linolenic acid production from RM18 in TWH accounted for 5.5% of total lipids, which was 1.58 times more than that of the control strain. Transcriptomic profiling indicated that enhanced central metabolism and terpene biosynthesis led to improved carotenoids production, whereas aromatic amino acid synthesis and DNA damage checkpoint and sensing were probably relevant to tea waste hydrolysate tolerance.
Conclusion:Tea waste is suitable for the hydrolysis of microbial cell culture mediums. The R. toruloides mutant RM18 showed considerable carotenoids and lipid production cultured in tea waste hydrolysate, which makes it viable for industrial applications.
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